Post-transcriptional RNA modification is quite complex as there are more than sixty known RNA modifications that occur in thousands of RNAs in eukaryotic cells. A number of these RNA modifications have been shown to influence the development and physiology of the central nervous system (CNS) and to be involved in carcinogenesis. Despite the growing interest in and importance of RNA modifications, the available tools that scientists have to monitor modified RNAs are limited. The goal of this contract is to generate a set of monoclonal antibodies for monitoring modified eukaryotic RNA, including messenger RNA and regulatory RNA. We propose to use a novel high throughput approach that utilizes the advantages of animal immunization and in vitro antibody selection to develop monoclonal antibodies highly specific to RNA modifications. To achieve proof-of-principle in Phase I, monoclonal single domain camelid VHH antibodies to four (4) RNA modifications, Inosine (I), 5-methylcytidine (m5C), N6-methyladenosine (m6A) and pseudouridine (?) nucleosides will be developed. In phase II, the effort will be dedicated to scale up production of monoclonal antibodies developed in phase I for commercialization, and expand the antibody development to 20 additional RNA modification targets. Availability of such reagents will enable researchers to monitor the effect of specific RNA modifications on the structure or function of respective individual RNA, or to analyze tissue sections, e.g. tumor samples, for the occurrence of such modifications.